Enzymes are proteinaceous catalytic materials that have great industrial potential. Enzymes also are often very expensive materials. They are generally soluble in their respective substrates and except where the conversion product is of great value, recovery of the enzyme for reuse may be difficult or impossible. In some cases, the processing conditions may destroy the enzyme. Where the enzyme is not destroyed, it may be necessary to destroy it, as in some food products, where continued activity would have an unwanted effect.
To avoid these problems, fixed or immobilized enzyme systems have been developed in recent years. Procedures such as adsorption, encapsulation, and covalent bonding are routinely used with many enzymes. The immobilization procedure selected, from the many available, produces a composition that can be used in either batch or continuous processes, but that is most advantageously used in a continuous process for economy.
While the term "insolubilized enzyme" has been used in the past on occasion, as in U.S. Pat. No. 3,519,538, to refer to an enzyme coupled by covalent chemical bonds to an insoluble inorganic carrier, and thus rendered not soluble in water, the term "immobilized" is used herein to refer to such an enzyme, or other biologically active material, fixed to a carrier.
The term "stabilized" is used herein to refer to a biologically active material, such as an enzyme, that has been stabilized against the loss of activity that would otherwise occur because of aging or exposure to an elevated temperature, or use in a reaction as a catalyst.
In the process of immobilizing an enzyme, there are many important practical considerations. There should be as little loss of enzyme activity as possible. The cost of immobilization should be low. The carrier material should be one that does not have a deleterious effect on the action of the enzyme during the process in which it is to be used. The immobilized enzyme should not leak enzyme or any other material into the reaction mixture, especially in food processing applications. The activity of the enzyme should remain high over a long period of operating (reaction) time, generally measured, in industrial processes, as the half-life. In addition, the immobilized enzyme should offer good hydraulic characteristics, to permit reasonable throughput rates. Equally importantly, the immobilized enzyme should be able to withstand reasonable operating temperatures, to permit practical operating rates, with the least feasible loss of activity.
For economy, it is also desirable that recharging of the carrier be possible, to reactivate spent immobilized enzyme, preferably by as simple an operation as possible.
Work in the field has progressed from concern simply with trying to immobilize an enzyme on a water-insoluble carrier to more sophisticated work in which the objective was to produce an immobilized enzyme that would deal successfully with all of the practical considerations mentioned above.
Several United States patents describe advances in the art that are representative of what has been done.
In U.S. Pat. No. 3,519,538, Messing and Weetall describe an immobilized enzyme composition in which the enzyme is covalently coupled to an inorganic carrier through an intermediate silane coupling agent, the silicon portion of the coupling agent being attached to the carrier and the organic portion of the coupling agent being attached to the enzyme. While glass of controlled porosity was the preferred carrier material, a wide variety of inorganic carrier materials, often siliceous, are disclosed as being useful. The carrier was prepared for use by substantial exposure to nitric acid, followed by furnacing in an oxygen atmosphere.
In almost all of the instances in which a particulate, siliceous carrier material is employed for initial reaction with a organosilane, the carrier particles are intially activated so as to present, at their surfaces, either oxygen or hydroxyl groups or both. The hydroxyl groups ordinarily are produced by reaction of the siliceous particles with a strong acid. Thus, Messing and Weetall in U.S. Pat. No. 3,519,538 washed powdered porous silica glass in 0.2 N nitric acid at 80.degree. C. with continuous sonication for at least three hours. The glass was then washed several times with distilled water, and then heated to 625.degree. C. overnight in the presence of oxygen. The treated glass was then considered to be ready for reaction with gamma-aminopropyltriethyoxysilane. Trypsin was coupled to the silated glass through the use of a covalent coupling agent.
In U.S. Pat. No. 3,556,945, Messing used an enzyme having available amine groups, and it was coupled to a porous glass carrier through reactive silanol groups, by means of amine-silicate bonds and by hydrogen bonding. Once again, the carrier was prepared for use by an acid treatment, coupled with heating in a furnace.
In U.S. Pat. No. 3,669,841, Miller describes immobilized enzyme compositions in which the enzyme is attached to siliceous materials by silation of the carrier, and linking to an enzyme by a crosslinking agent. No pretreatment of the carrier particles is mentioned. In Example 1 of the patent, gamma-aminopropyltrimethoxysilane is reacted with particulate silica, then an enzyme is added with stirring, and then an aqueous formaldehyde solution is added. In Example 3, a calcium silicate carrier, in a form not specified, was "dry coupled with 0.25 weight percent gamma-aminopropyltrimethoxysilane, was slurried in water (50 ml.) and B. subtilis enzyme mixture" was added to the slurry. The pH of the slurry was adjusted to 8.0 and glutaraldehyde was added as a covalent coupling agent. At the point in the example where the pH was alkaline, the calcium silicate carrier had already been reacted with the organosilane.